Lung endothelial cells regulate pulmonary fibrosis through FOXF1/R-Ras signaling

Pulmonary fibrosis results from dysregulated lung repair and involves multiple cell types. The role of endothelial cells (EC) in lung fibrosis is poorly understood. Using single cell RNA-sequencing we identified endothelial transcription factors involved in lung fibrogenesis, including FOXF1, SMAD6, ETV6 and LEF1. Focusing on FOXF1, we found that FOXF1 is decreased in EC within human idiopathic pulmonary fibrosis (IPF) and mouse bleomycin-injured lungs. Endothelial-specific Foxf1 inhibition in mice increased collagen depositions, promoted lung inflammation, and impaired R-Ras signaling. In vitro, FOXF1-deficient EC increased proliferation, invasion and activation of human lung fibroblasts, and stimulated macrophage migration by secreting IL-6, TNFα, CCL2 and CXCL1. FOXF1 inhibited TNFα and CCL2 through direct transcriptional activation of Rras gene promoter. Transgenic overexpression or endothelial-specific nanoparticle delivery of Foxf1 cDNA decreased pulmonary fibrosis in bleomycin-injured mice. Nanoparticle delivery of FOXF1 cDNA can be considered for future therapies in IPF.


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March 2021

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All the raw sequence data generated in this study is deposited in GEO database (accession number GSE213018; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc=GSE213018). Single cell RNA sequencing data generated in this study from donor and IPF patient lungs (Supplemental Figure S4) were deposited in GEO database (accession number GSE213017). Single cell RNA sequencing data generated in this study from bleomycin-treated and untreated adult mice lung were deposited in GEO database (accession number GSE213016). Single cell RNA sequencing data of Northwestern University (NW) datasets from donor and IPF lungs were retrieved from GEO database (GSE122960; https://www.ncbi.nlm.nih.gov/geo/). Bulk RNA sequencing data generated in this study from bleomycin-treated control and endFoxf1+/-mouse lung endothelial cells were also deposited in GEO database (GSM6578251 and GSM6578252). Source data are provided with this paper.
Both sexes were used. We have not noticed any sex-dependent differences.

NA
The Institutional Review Board of the Cincinnati Children's Hospital Medical Center (Federalwide Assurance #00002988) approved all the studies with human tissue samples (IRB protocol #2017-4321). Human lung tissue specimens were obtained from tissue repository at University of Cincinnati Medical Center that provides de-identified human biospecimen procurement and banking services in support of basic, translational, and clinical research. Total number of ten lung tissue samples were received, including six males and four females. All patients signed informed consent prior to tissues collection. Explanted lungs were acquired from donors with end-stage IPF lung disease undergoing transplant or from rejected control donor lungs.
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For the in vivo studies, no formal randomization method was used as the genotype of the mice was known before starting the experiment. Mice were randomly distributed in different cages for the entire duration of the experiment. For in vitro studies, samples were randomly allocated into experimental groups.

March 2021
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